Human cancer and in vitro transformation are thought to arise after multiple progressive changes in gene expression. By the time a cell is fully transformed, several percent of gene transcripts are newly expressed and several percent are lost. If one could capture cells committed to transformation, but prior to any phenotypic alteration, then one might be able to understand the earliest events in this process. C3H 10T1/2 cells are widely used in transformation studies, because of their extremely low background spontaneous transformation frequency, and their transformability by a wide variety of agents. The DNA demethylating drug 5-aza-2'- deoxycytidine transforms these cells at a surprisingly high frequency (2-3%) and at long latency (4-6 weeks), after a 24 hour exposure to the drug. These properties have permitted development of a novel strategy for studying early transformation. Individual cells are plated in microwell dishes, treated for 24 hours, and then allowed to grow to subconfluence, at which time each micro- colony is subdivided for replating and simultaneous cryopreservation. Using this strategy, ancestral cells committed to transformation are captured prior to any phenotypic change. These pretransformed cells will be studied in several ways. cDNA libraries will be constructed from pretransformants and screened using RNA from transformed, pre-transformed, and nontransformed cells, in order to identify genes expressed prior to phenotypic transformation. DNA from pretransformants will be transfected into untreated cells, in order to identify dominantly acting pretransforming genes. Chromosomes of pretransformants will be karyotyped in order to identify deletions or rearrangements. Finally, we will determine the nature of the early pretransformation genes, in order to determine whether DNA methylation or other mechanisms are involved.